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This laboratory is interested in chromatin regulated gene expression with an emphasis on innate immunity. We currently focus on two nuclear regulatory factors Brd4 and IRF-8. Brd4 is a mammalian bromodomain protein that interacts with acetylated chromatin. IRF-8 is a DNA binding transcription factor of the IRF family important for innate immunity. Our goal is to elucidate the mechanism by which these two factors act on chromatin and regulate transcription in living cells.
IRF-8 binds to chromatin by interacting with partner proteins in living macrophages
Dendritic cells (DC) and macrophages are the main cell types that confer innate immunity upon infection with pathogens. Previous studies from this laboratory showed that IRF-8 plays an essential role in the development of macrophages and establishment of innate immunity. We recently found that IRF-8 is also important for the development and function of DCs. Further analysis showed that IRF-8 and a related IRF protein, IRF-4 regulate the development of the entire DC subsets. That is, IRF-4 knock-out (KO) mice lack CD4+ DCs, while IRF-8 KO mice lack CD8 a + DCs and pDCs. Accordingly double KO mice have hardly any DCs . Retroviral transfer of IRF-4 and IRF-8 into double KO bone marrow cells rescued expression nof a series of genes specific for corresponding DC subsets. These data establish that IRF-4 and IRF-8 are the basic backbone of DC development. We also found that IRF-8, but not IRF-4 rescues Type I interferon, and IL-12 expression in double KO DCs, indicating that IRF-8 is an obligatory factor for the elicitation of innate immunity.
To study binding of IRF-8 to chromatin in live macrophages, we have employed the fluorescence recovery after photobleaching method. This method allows us to visualize dynamic movement of transcription factors and their real time interactions with chromatin in living cells. We found that the majority of IRF-8 is moving extremely fast, binding to chromatin for less than 0.1 second and then moving away to bind again elsewhere. Only about 10% of IRF-8 showed more stable interaction. A macrophage activating stimulus (IFN g /LPS) markedly increased the more stably bound fraction, indicating that an immunological stimulus can change a global behavior of IRF-8. We found that interaction of IRF-8 with chromatin was contingent upon its interaction with the partners, PU.1 and IRF-1. Together, these results are consistent with the idea that transcription factors are interact with chromatin only transiently, constantly scanning the entire genome. Live cell technologies such as photobleaching open a new horizon to our understanding of nuclear events and gene expression.
Figure 1. IRF8-GFP induced macrophage differentiation in Tot2 progenitor cells, but the mutant IRF8-GFP did not. FRAP analysis showed that wild type IRF8 binds to chromatin with characteristic fast on-and-off kinetics, but the mutants showed no sign of chromatin binding.
Brd4 and epigenetic memory
Brd4 is a mammalian bromodomain protein whose function has remained obscure. We have previously shown that it preferentially binds to acetylated core histones, H3 and H4 in living cells. Brd4 also associates with chromatin during mitosis, a feature unusual for a nuclear regulatory protein. Another structurally related factor, Brd2 also recognizes acetylated chromatin and associates with mitotic chromosomes. Based on these features Brd2 and Brd4 are thought to play a role in the inheritance of epigenetic memory. To gain insight into the role of Brd4 in gene expression we searched for a non histone protein that interacts with Brd4. By immunopurification and mass-spectrometry analysis we identified P-TEFb as a stable partner of Brd4. P-TEFb forms the cyclinT/Cdk9 heterodimer and phosphorylates the C-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation of nascent RNA. The Brd4/P-TEFb complexes we identified are novel and differed from the previously known P-TEFb complex that is bound to the inhibitory subunit. By a series of functional studies we found that the Brd4 bound P-TEFb complex is functionally active, competent in CTD phosphorylation and is capable of stimulating transcription. Analysis of HIV-1 LTR promoter activity combined with chromatin immunoprecipitation assays led to a model in which Brd4 recruits P-TEFb to the promoter in acetylated chromatin to stimulate transcription. This work is extended to a more recent microarray analysis to compile cellular genes controlled by Brd4/P-TEFb. In these studies we have used the siRNA approach and examined cells in which Brd4 was under-expressed. Our current goal is to address how transcriptional memory may be transmitted through Brd4 across cell division.
Dey et al
Hoechst : Anti-Brd4 Ab
Figure 2. Brd4 and Brd2 belong to the BET family and have two bromodomains through which they interact with acetylated chromatin. They remain associated with chromatin during mitosis. The lower panel shows immunostaining of endogenous Brd4 on spread chromosomes.
Figure 3. An equilibrium model for Brd4-P/TEFb interactions. P-TEFb occurs either complexed with Brd4 or the inhibitory subunit. The Brd4 bound P-TEFb is recruited to a promoter in acetylated chromatin and stimulates RNA polymerase II dependent transcription.